3D生物打印的肝结构体植入兔纤维化肝的研究

Study on 3D Bioprinted Liver Structure Implanted in the Fibrosis Liver of Rabbits

通过将三维(3D)生物打印的类肝水凝胶结构体,经肝表面贴覆法植入纤维化肝兔模型,探讨改善肝纤维化以及形成类肝组织结构的可行性。采用兔离体肝脏胶原酶消化法提取兔原代肝细胞,并采用四氯化碳(CC14)皮下注射诱导兔肝纤维化模型。选取30只肝纤维化兔,随机分为实验组、对照组、假手术组,每组10只。3D生物打印兔肝细胞-海藻酸钠-明胶3D水凝胶网格状结构体片段,经肝表面贴覆法植入兔纤维化肝,为实验组。植入16 d后,检测兔肝功能生化指标及肝脏组织病理学变化,观察肝纤维化的发展程度,以及类肝组织结构形成情况。按照设计的参数,打印出含有兔原代肝细胞的网格状水凝胶结构体。通过LIVE/DEAD双荧光染色观察,打印后肝结构体的细胞存活率为82%±3%。兔肝细胞水凝胶支架体内植入16天后,可见每个植入物的大部分支架材料尚未降解,未降解的植入物与肝脏贴合紧密,融合生长。通过检测兔肝功能生化指标及植入物之下的肝脏组织病理学观察,实验组兔肝功能生化指标分别为:ALT(90.26±13.05)U/L,AST(75.37±13.45)U/L,γGT(16.62±6.72)U/L,ALB(32.48±4.43)g/L,与对照组、假手术组之间比较均P>0.05。实验组、对照组、假手术组的肝纤维化程度病理学检查评分结果分别为2.95±0.50、3.11±0.58、3.02±0.62,各组之间比较均P>0.05。兔肝细胞-3D水凝胶支架的植入,使肝功能生化指标及兔肝脏纤维化程度稍有改善,但各组之间比较均无统计学意义。组织学观察结果表明,实验组植入物中肝细胞均匀分布,未见细胞变性及死亡,可形成类肝组织结构。3D生物打印的肝结构体具有再造肝样组织片段和实现部分肝功能的潜力,将在基础医学和临床之间搭建桥梁,为肝脏再生打下基础。

Three dimensional(3 D) bioprinted liver-like hydrogel structures were implanted into the fibrosis liver of rabbit models by the liver surface cladding method to study its feasibility of improving the liver fibrosis and of generating liver-like tissue. Primary hepatocytes were extracted from the liver tissue of rabbit by 0.1% collagenase type IV digestion, and the hepatic fibrosis model was established by subcutaneous injection of carbon tetrachloride(CC14) into rabbits. Thirty rabbits with liver fibrosis were randomly divided into experimental group, control group, and sham operation group, with 10 rabbits in each group. The structure lamella containing rabbit primary hepatocytes/alginate/gelatin blend was constructed using 3 D bioprinting technique. Then the 3 D structure lamellas were implanted into the liver surface of hepatic fibrosis rabbits, as an experimental group. Sixteen days after implantation, liver functional biochemical index and liver histopathological changes of the liver were examined as well as the developmental degrees of hepatic fibrosis and the formation of liver-like tissue structure. The 3 D structures with the grid hydrogel composing of multilayer staggered cylindrical microfilament were obtained and live/dead cell fluorescent staining results showed that the hepatocytes’ survival rate was about 82% ± 3% after printing. Most of the implants were not degraded 16 days after the implantation. The undegraded implants were tightly integrated into the liver. The biochemical indexes of liver function in the experimental group were ALT(90.26±13.05)U/L, AST(75.37±13.45)U/L, γGT(16.62±6.72)U/L, ALB(32.48±4.43)g/L. The significance between any two groups was P>0.05. The pathological examination scores of liver fibrosis degree in the experimental group, control group and sham operation group were 2.95±0.50, 3.11±0.58 and 3.02±0.62, respectively, and the significance between any two groups was P>0.05. The results showed that the fibrosis degree and the function of the rabbits’ liver had improvements to some extents in experimental group though there was no statistical significance. Histological observations from the experimental group showed that hepatocytes were evenly distributed, and that cell degeneration and death were not discovered, succeeding in generating liver-like tissue structure. In conclusion, the bioprinted hepatic structure has potentials of regenerating liver-like tissue structure and rebuilding liver functions.